The major long term objective of our work is to gain insight on the mechanisms of how erythropoietin (EP) regulates erythroid development in murine and human systems. EP is essential for the continued of early erythroblasts and colony forming unit-erythroid (CFU-E) cells. When EP is withdrawn from these cells, rapid apoptosis of these cells takes place within 24 h period (Koury and Bondurant, 1990). It has been demonstrated that apoptosis in other eukaryotic cell systems is regulated by the action of the Bcl-2 and ICE cysteine protease gene families. In the preliminary work, I have demonstrated the presence of several members of the Bcl-2 family with Bcl-X(long) undergoing a pronounced upregulation in the presence of EP. have also demonstrated the rapid activation of a specific member of the ICE cysteine protease family, YAMA/CPP32/apopain(apopain) in erythroid cells deprived of BP. There are two specific aims of this research proposal. The first aim is to evaluate he role of Bcl-2 family members (specifically Bcl-X, Bax, and Bad) in control of apoptosis in erythroid development. This will done by: 1) determining the stoichiometric ratios between these proteins by immunoprecipitation and quantification using known amounts of standards on acrylamide gels 2) evaluating whether or not Bax is present only as heterodimers with Bcl-X, by use of immunoprecipitation, resolution on SDS-PAGE gels and Western blotting 3) determining the intracellular location of Bcl-X and seeing if it persists in reticulocytes by visual cytoimmunostaining and immunoelectron microscopy, and 4) identifying what bcl-x mRNAs are expressed in the erythroid cells during development by the use of RT-PCR to discriminate tween differentially spliced forms of bcl-x mRNA The second specific aim is the analysis of (See Part 21 Continuation Page)